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Figure 1 Analysis of a mixture of 7 sulfonamides with diode array at 10 pg and 100 pg absolute with 100-nL injection volume.
MS conditions Source: ESI Ionization mode: positive Vcap: 4000 V Nebulizer: 10 psig Drying gas flow: 7 l/min Drying gas temperature: 150 C SIM ions: 0 min: 172.9 (sulfanolamide) and 215.1 (sulfaguanidine), 3.6 min: 214.9 (sulfacetamide), and 251.1 (sulfadiazine), 5.6 min: 250.1 (sulfapyridine), 279.1 (sulfamethazine) and 281.1
Results Figure 1 shows the analysis of seven sulfonamides with 100 pg and 10 pg on-column with diode-array detection. With a signal-to-noise ratio of 3, the 10 pg injection is at the detection limit. Figure 2A shows the MS signal (total ion chromatogram) for the same analysis. The injected amounts for MS are 10 pg and 1 pg (10 times less than with DAD). Several of the sulfonamides elute closely and extracting just the ion of interest allows for better integration and calibration, especially at low levels. In addition, using extracted ion chromatograms (EICs) can enhance the visibility of smaller components in a mixture. Figure 2B illustrates the use of EICs for three of the components of the sulfonamide mixture at 1 pg on-column. Relative standard deviation for the sulfonamides was < 0.1 % for retention time and < 2.8 % for area respectively, based on ten runs with 100 pg and 100 nl injection volumes (table 1). The assay was linear in a range from 1 pg/l to 1 ng/l (r2 >0.9999) for the MSD and 10 pg/l - 100 ng/l for DAD. Even when dealing with extremely small sample volumes, the capillary LC system is capable of giving highly reproducible quantitative results and which makes the system ideal in situations where sample volumes are limited and multiple analyses are necessary on a single sample. A combined capillary LC/MS system enhances the sensitivity and specificity of the analysis.
Sulfameter Run # Mean S.D RSD RetTime [min] 8.083 4.69e-3 0.058 Sulfameter Mean S.D. RSD 8.346 4.54e-3 0.054 Signal: DAD1 A, Sig=278,20 Ref=450,80 Amount [pg} 118.6 2.01 1.70 Area [mAU*s] 21.96 0.35 1.62 Height [mAU] 3.41 0.044 1.29 Width 0.099 8 e-4 0.857 Symm.[min] 0.83 0.01 1.70
Equipment
Agilent 1100 Capillary LC system consisting of Capillary pump Micro vacuum degasser Thermostatted column compartment Micro autosampler Diode array detector, 500 nL flow cell Agilent ChemStation Agilent 1100 Series LC/MSD SL Electrospray source Capillary LC nebulizer Column ZORBAX SB C-18, 150 0.5 mm, 3.5 m (Agilent p/n 5064-8262)
Signal: MSD1 TIC, MS File) 116.9 1.87 1.60 2.46e5 3943 1.60 2.47e4 358 1.44 0.1495 3.40e-3 2.27 0.81 0.01 1.46
B
1 2 3 4 sulfaguanidine sulfanilamide sulfacetamide sulfadiazine 5 6 7 8 sulfapyridine sulfamerazine sulfamethazine sufameter
2500 2200 2200 sulfamethazine m/z 279.1 sulfacetamide m/z 215.1
A
Abundance 5000
7 1 5 6 2 3 10 pg 0 1 pg
2
600 1600
400
Rainer Schuster is an application chemist at Agilent Technologies, Waldbronn, Germany. Christine Miller is an application chemist at Agilent Technologies in Palo Alto, CA For more information on our products and services, visit our worldwide website at http://www.agilent.com/chem
10 min
Figure 2 Analysis of the standard mix with 10 pg and 1 pg with MSD and SIM - total ion chromatogram from SIM (A) and extracted ion chromatogram from SIM(B)